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� Physico-chemical imobilisation
� Adsorption
� Entrapment
� Cross-linking
� Chemical modification
� Self assembling
� Covalent modification
� Affinity assembling
Of what we will talk?
Main elements of a biosensor.
(a) Biorecognition element
(b) transducer
(c) amplifier
(d) Signal converter
(e) recorder
Popular approaches for the immobilisation of biorecognition elements
• Adsorption
• Entrapment
• Crosslinking
• Encapsulation
• Covalent binding
Bio-molecules immobilisation
Easy to perform
Complexto perform and
time consuming
Physico-chemical Adsorption
Surface self assembling of bio-molecules Adsorption
Advantages: Easy and cost effective approach, less destruptive (biorecognition element mantain their function.
Problems: low controll of molecules orientation, less stable.
Forces governing adsorption
Limitations
+ +- -
Poor stability of the modified layer
Poor orientation of the biorecognition elements
Entrapment of biomolecules
• Membranes -Enzyme membranes can be prepared by attaching enzymes to
membrane-type carriers, or by molding into membrane form.
Example is the entrapping of enzymes in nafion membrane.
Electrostatic self assembly
Palyelectrolyte –The enzymes are trapped in between sequencial layers of charged polymers.
Electrostatic self assembly – protein
multilayers
Colorimetric detection of Glucose via the use of Sequential Enzymatic Reaction
Glucose oxidase
Peroxidase
DA67: Chromogenfor enzymatic reaction transduction
Entrapping biomolecules by electropolymerisation
Possible monomers
Entrapping in hydrogels
Hydrogel: is a network of polymer chains that are hydrophilic; this contains high quantity of water.
Cross-linking
Cross-linking of the enzymes ensure higher stabilityand robustness to the sensor.
Cross-linking is obtained by using molecules that are able to interact with the aminoacidic residues and to bridge two neigbouring enzymes..
Covalent binding
Chemical modification
Advantages:
• More stable
• Allow immobilisation to a wider range of surfaces (e.g. metal, semi-conductor, polymer etc.).
• Allow to immobilise biomolecules in a more controlled way (better orientation, better reproducibility).
• Allow to create suitable environment for:
- Biomolecules immobilisation
- Reduction of non-specific interaction
Disadvantages:
• Required chemical modification of the biomolecules.
• Harsh chemical reaction may affect the function of the biomolecules.
• Time consuming and increased complexity in sensor preparation.
Metal surface functionalisationSelf assembling monolayer (SAM)
Spontaneous but ordered assembling of molecules.
Most used is the self assembling of alkane thiols(R-SH).
This approach allow to create stable intermediate layer for the controlled immobilisation of biomolecules onto metals (Au, Cu, Ag)…
Si Self-Assembled Monolayers (SAMs)
Equivalent to Thiol SAM but:
More stableSuitable for glass, quartz, Pt and other semiconductors where thiols do not attach.
Polymeric substrate
Electrode
Covalent Binding
The covalent binding method is based on the binding of biomolecules by covalent bonds. The functional groups that may take part in this binding are listed below:
Amino group; Carboxyl group; Sulfhydryl group; Hydroxyl group; Imidazole group; Phenolic group; Thiol group; Threonine group; Indole group.
Covalent attachment to a support matrix must involve only functional groups of the biomolecule that are not essential for catalytic/recognition activity.
Functional group of proteins
Covalent binding (EDC/NHS)
Bio-affinity assembling
Streptavidin: is a 60000 dalton protein purified from the bacterium Streptomyces avidinii. Its homo-tetramers have an extraordinarily high affinity for biotin. Dissociation constant (Kd) on the order of ≈10-14 mol/L.
Biotin: also known as Vitamin H or Coenzyme R, is a water-soluble B-complex vitamin (vitamin B7).
Schematic representations of IgG
immobilization on a gold surface
modified by various cysteine-tagged
protein G constructs. Anal. Chem.,
79 (7), 2680 -2687, 2007.
Schematic representations of
DNA-directed antibody
immobilization by protein G-
DNA conjugate. Anal. Chem.,
79 (17), 6534 -6541, 2007
(a) Fluorescence response to different concentrations of CA 19-9 antigen (b) Schematic illustration of the
sandwich immunoassay configuration used. (c) Fluorescence intensity as a function of the CA 19-9 concentration
in the samples. (d) Specificity of the antibody array. Antibodies for CA 19-9 and CEA were spotted onto the array,
and samples containing either CA 19-9 or CEA were added. J. Am. Chem. Soc., 128 (3), 676 -677, 2006
Supramolecular approach
Summary
• Different approaches for the immobilisation of the biorecognitionelement onto the transducer are possible.
• Degree of complexity increases with the increase in the control of the final surface.
• Chemical, biological and supramolecular interaction can be used to functionalise surfaces.
• Recognition ability of biorecognition element can strongly influenced by the immobilisation process.
